Alpha-lactalbumin (LA) and lysozyme (LZ) are homologous proteins with closely similar 3D structures but highly divergent functions. LA, the regulatory protein of lactose synthase, modulates the specificity of the catalytic component, galactosyltransferase (GT) so that it can catalyze the biosynthesis of lactose, whereas LZ catalyzes the hydrolysis of glycosidic bonds in bacterial cell walls. LA also binds a Ca2+ ion with high affinity. The Ca2+ is not required for activity and, because the activity of an LA molecule occurs briefly during its passage through an intracellular compartment, is unlikely to be necessary for stabilization. Ca2+ is, however, required for regenerating native LA from the reduced denatured form. Although the majority of known LZs do not bind Ca2+, a subclass has been recently identified that do bind Ca2+, apparently at a site corresponding to that in LA. Having expressed that cDNA for LA in E. coli as a fusion protein from which native active LA can be prepared, we propose to (i) use site-directed mutagenesis to test hypotheses regarding structure-function relationships in LA, and the structural basis of its functional divergence from LZ; (ii) continue investigations of structure and action in GT using chemical procedures. Unique crosslinking sites will be introduced into LA to facilitate the identification of the binding site for LA on GT; (iii) conduct further refolding studies with LA and Ca2+- binding LZs from the reduced denatured state to determine if the renaturation of LA is Ca2+-dependent over a wider range of conditions and to elucidate the folding properties of these LZs; (iv) change aspartyl residues in the Ca2+-binding site of LA to determine if the coupling of native folding with Ca2+ binding is a function of the charge distribution in the Ca2+-binding elbow; (v) characterize the physical properties and stabilities of mutant LAs generated in (i) and (iv). For mutants with interesting properties this will involve a collaborative study of their 3D structures by X-ray crystallography; (vi) express the cDNA for chicken LZ and to determine the functional and structural effects of LA-like sequence changes. This is a long-term goal that is dependent on results obtained in (i) and (iv).